Virginia Miller, PhD

Molecular and genetic analysis of virulence of Yersinia and Klebsiella: My laboratory uses Yersinia, and Klebsiella as model systems to study bacterial pathogenesis. The long-term goals of our work are to understand the bacteria-host interaction at the molecular level to learn how this interaction affects the pathogenesis of infections and to understand how these pathogens co-ordinate the expression of virulence determinants during an infection. To do this we use genetic, molecular and immunological approaches, in conjunction with the mouse model of infection. Some of our projects are outlined below.

RovA regulon of the Yersiniae

We have applied several genetic approaches to identify new virulence genes of Y. enterocolitica and are currently characterizing these genes, their products, and their role in disease. We also have been studying the invasion gene inv, with a focus on understanding the mechanism of regulation of expression of inv and the co-ordination of its expression with other virulence genes. An inv regulatory gene, rovA, has been identified that regulates expression of inv in the laboratory and during an infection. RovA also regulates expression of other novel virulence determinants that influence the early inflammatory response to Y. enterocolitica infection. Recently we extended the analysis of RovA to Yersinia pestis, the causative agent of bubonic and pneumonic plague and found that RovA is also required for full virulence of Y. pestis. Microarray analysis was used to identify the RovA regulated genes in Y. pestis and Y. enterocolitica. Sixty-four genes appeared to be RovA regulated in Y. enterocolitica and 73 in Y. pestis, suggesting the regulon may be quite large in each of these species. Our long-term goals are (i) to understand how these genes are regulated by RovA and how that is coordinated with expression of other virulence factors, and (ii) to determine which RovA regulated genes contribute to virulence and understand how they affect the host-pathogen interaction.

Ysa Type Three Secretion System of Y. enterocolitica

Type III secretion systems (T3SS) are a means by which Gram negative pathogens deliver effector proteins into host cells. One of the first and best, characterized systems is encoded on the virulence plasmid of the yersiniae. However, a second T3SS was recently identified on the chromosome of Y. enterocolitica (designated the Ysa T3SS). We identified some of the key players in the regulation of expression of this system and have begun to identify the effectors (designated Ysps) secreted by the system. The long-term goals are to understand the role of this system and the individual effectors in the biology of Y. enterocolitica as well as its regulation. However, the current focus of our research is on the regulation of this system. Our data indicates that the expression of the Ysa T3SS is dependent on a phosphorelay system with a number of unusual features. Typically in bacteria these phosphorelay systems are composed of two proteins (i.e. the two component regulatory system) in which one protein is the sensor (responds to an environmental signal) and the other the response regulator (usually a DNA binding protein). Some related but more complex systems (termed hybrid two component systems) have several transfers of phosphate in the sensor kinase component before the ultimate transfer to the response regulator by the histidine phosphotransferase (Hpt) domain of the sensor. What makes the regulation of the Ysa T3SS system unusual is that the Hpt “domain” (YsrT) is a separate, small protein rather than a domain of the sensor (YsrS). In addition, a second response regulator, RcsB, from another two-component system is required in addition to the response regulator YsrR for ysa expression, even though the sensor of the Rcs system is not required. How exactly these players are functioning to regulate the ysa/ysp system and the consequences of the unique regulatory set-up is the topic of ongoing research.

Autotransporter proteins of Y. pestis

A group of proteins that have a high likelihood of contributing to Y. pestis pathogenesis/life cycle either in the mammalian or flea host are the autotransporter proteins (ATs). Classic ATs (also known as Type Va secreted proteins) consist of three basic domains: a N-terminal signal sequence, a “passenger domain” of variable size and finally a ß -domain of 250-300 amino acids at the C-terminus which facilitates translocation of the passenger domain (PD) across the outer membrane. The PD provides the functional activity of the protein (adhesin, cytotoxin, protease, etc) and new activities are being attributed to these proteins as more of them are studied in detail. While the ß -domains are relatively conserved between ATs (allowing identification of an AT in the genome) the PDs can vary significantly. ATs represent the largest class of secreted proteins in Gram-negative bacteria with now more than 1000 identifiable in sequenced genomes. Despite this, only a relatively small number have been studied in any detail and many questions remain as to the full range of functions that can be performed by these diverse proteins. In silico analyses have identified ten Type Va AT proteins of Y. pestis (designated yaps/Yaps). As most ATs that have been studied in other organisms have functions associated with virulence and these proteins are either surface localized or secreted these are attractive for study because they have a high probability of playing a role in Y. pestis pathogenesis and also represent attractive potential vaccine or therapeutic targets. The Yaps of Y. pestis are also of interest to pursue because they do not show significant similarity to the better studied ATs and thus may possess novel functions. Furthermore, the availability of an excellent animal model (mice are a natural host for Y. pestis) for studying the functional and biological role of these AT proteins, combined with the ability to do molecular genetics in Y. pestis, makes this an excellent experimental system overall. We have preliminary data indicating that all ten of the yaps are expressed not only under standard laboratory conditions but also during infection; indeed, some are highly induced during infection. We also have begun analyses of the localization of the Yaps and found that while some appear to be localized and exposed on the bacterial surface, others appear to be released into the culture supernatant. In addition, we have constructed in-frame deletion mutants in eight of the ten yaps in a fully virulent Y. pestis strain background and have begun testing the effect of these mutations on virulence. While these tests are ongoing, four of these mutants clearly have phenotypes in a bubonic plague model of infection (pneumonic plague has not yet been tested). These results are consistent with our hypothesis that the yaps play a role in pathogenesis. Our long-term goals are to understand at a molecular level the role(s) of the individual Yaps in disease. Currently we are (i) studying the localization and expression of the Yaps; (ii) continuing our analysis of the role of Yaps in the virulence of Y. pestis; (iii) investigating the potential functions of the Yaps.

Virulence factors of Klebsiella

For Klebsiella we have developed a mouse model of infection using an intranasal inoculation method. A bank of 5,000 transposon mutants have been isolated and screened in this intranasal model of infection for mutations that alter the ability of Klebsiella to either colonize the lung or spread from the lung to the spleen. A subset of these mutants is currently being studied in more detail. In particular we are interested in two mutants that identify a putative Type VI secretion system (T6SS) of Klebsiella. The T6SS have emerged in recent years as a new type of secretion system in Gram-negative bacteria and has been linked to virulence in both mammalian and plant pathogens.